This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Electron tomography of rapidly frozen, freeze-substitution fixed samples will be used to examine a significant fraction of the volume of metaphase spindles from U2OS cells, followed by comparable work on cells frozen and fixed during spindle formation. Our structural studies will employ improved methods for sample preparation (TR&D 3A) and both large-scale montages and multiple serial sections, assembled into 3D reconstructions (TR&D 5D). The trajectories of spindle microtubules (MTs) will initially be determined by hand modeling, though in subsequent work we hope to have software for automatic segmentation. The resulting models of MT trajectories will be studied with cluster analysis and several application-specific software tools to compare MT organization with the predictions from four distinct and popular models for spindle formation: the classic, centrosome-initiation model, the idea that spindle MTs self-organize as a result of MT-associated motor enzymes, the idea of MT initiation and/or stabilization in response to gradients that emanate from chromosomes, and the concept that a non/MT "matrix" guides the formation and organization of MTs. The data should eliminate or at least erode one or more of these models and will certainly provide baseline structural information for the interpretation of functional experiments on spindle formation and function that are being carried out in our labs and elsewhere.